WNT/β-catenin regulatory roles on PD-(L)1 and immunotherapy responses

Dysregulation of WNT/β-catenin is a hallmark of many cancer types and a key mediator of metastasis in solid tumors. Overactive β-catenin signaling hampers dendritic cell (DC) recruitment, promotes CD8+ T cell exclusion and increases the population of regulatory T cells (Tregs). The activity of WNT/β-catenin also induces the expression of programmed death-ligand 1 (PD-L1) on tumor cells and promotes programmed death-1 (PD-1) upregulation. Increased activity of WNT/β-catenin signaling after anti-PD-1 therapy is indicative of a possible implication of this signaling in bypassing immune checkpoint inhibitor (ICI) therapy. This review is aimed at giving a comprehensive overview of the WNT/β-catenin regulatory roles on PD-1/PD-L1 axis in tumor immune ecosystem, discussing about key mechanistic events contributed to the WNT/β-catenin-mediated bypass of ICI therapy, and representing inhibitors of this signaling as promising combinatory regimen to go with anti-PD-(L)1 in cancer immunotherapy. Ideas presented in this review imply the synergistic efficacy of such combination therapy in rendering durable anti-tumor immunity.


Introduction
Wingless-related integration site (WNT)/β-catenin is an immunosuppressive signaling [1] that its activity in a tumor is indicative of low rates of immune infiltration [2].WNT/βcatenin signaling is a critical mediator of melanoma metastasis [3], orchestrating a T cell exclusion profile [4].Disruption of WNT/β-catenin signaling is reported as a strategy for suppression of invasion and metastasis in non-small cell lung cancer (NSCLC) [5].β-catenin activation shapes the immune desert landscape of hepatocellular carcinoma (HCC) [6].The suppressive effect of WNT/β-catenin on CCL4 contributed to the cold immune phenotype of melanoma [7].Mutations in the WNT/β-catenin occur in about 70% of microsatellite stable colorectal cancer (CRC) patients [8].The frequency of β-catenin + tumor cells and programmed death-ligand 1 (PD-L1) + immune cells can be regarded as an indicator of CRC progression [9].WNT/β-catenin activity promotes CRC progression through induction of epithelial-mesenchymal transition (EMT) [10].Expression of Frizzled-10 (Fzd-10) receptor and further β-catenin activation promote cancer stem cell (CSC) expansion and predicts weak prognosis in HCC [11].Enriched activity of this signaling in tumors with cold immunity (non-T cell-inflamed) provides a rationale for development of inhibitors in order to restore immune infiltration and increasing the efficacy of immunotherapy [12].There are signs of evidence indicating the combination of impact of WNT/β-catenin blockade with immune checkpoint inhibitors (ICIs) for better promotion of anti-tumor immunity against cancers like NSCLC [13] and melanoma [14].The aim of this review is to justify the mechanistic backbone of WNT/β-catenin-mediated ICI resistance, as well as rationalizing a possibility of the application of WNT/β-catenin blockade as a combinatory regimen with anti-PD-(L)1 aiming at a durable anti-cancer therapy.

Signaling elements
WNT (WNT5a) is a gene assessed to evaluate mesenchymal transition [15], and β-catenin is a critical mediator of WNT signaling [16].In fact, WNT proteins co-express to act synergistically for activation of β-catenin signaling in several cell types [17].Adenomatosis polyposis coli, CTNNB1 and AXIN (AXIN1 and AXIN2) are β-catenin signaling elements [12].Adenomatosis polyposis coli is a gene related to the suppression of WNT/β-catenin [18], which shows mutations in more than 90% of sporadic colon cancer cases [19].CTNNB1 is a gene encoding β-catenin that is mutated frequently in HCC.CTNNB1 mutation results in the cytoplasmic accumulation of β-catenin, which subsequently causes aberrant activation of WNT [20].AXIN is a cytoplasmic protein that acts as a negative regulator of WNT pathway and promotes β-catenin degradation [21].AXIN2 can be assessed as a marker for analyzing the activity of WNT pathway [22].WNT/βcatenin pathway signals via interaction with Fzd receptor family as well as different co-receptors [23].Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) is a co-receptor located on cell surface that is involved in the initiation of WNT/β-catenin pathway [18].Upon WNT activation, β-catenin degrading complex is inactivated, which results in β-catenin accumulation within the cytosol and its further stabilization.The stabilized β-catenin further translocated into the nucleus where it bonds to the T cell transcription factor (Tcf)/lymphoid enhancer-binding factor 1 (Lef1) [24,25].β-catenin is a Tcf1 transcriptional coactivator in which interactions within the β-catenin/Tcf1 axis are vital for transcriptional regulation.The Tcf1 long isoform contains β-catenin binding domain that mediates β-catenin recruitment to the protein complex [26].WNT is palmitoylated by porcupine (PORCN).PORCN activity is vital for secretion of WNT and its bondage to Fzd in responder cells [27].PORCN inhibition disrupts secretion of WNT and hampers stem cell activity in tumors [27], so it can be a target in WNT-driven cancers [28].Fzd receptors are other targets for WNT pathway suppression in human cancers [23].Dickkopf-related protein 1 (DKK1) is a known antagonist of WNT that acts through suppression of WNT interaction with Fzd receptors [29].Hindering the secretion of WNT ligands, interfering with interaction between WNT ligand and receptor, increasing the degradation of β-catenin or blocking interaction between β-catenin with its target genes are strategies for hampering WNT/ β-catenin signaling.Monoclonal antibodies against Fzd receptors, such as vantictumab (OMP-18R5), Fzd8 fusion proteins and extracellular traps for WNT ligand signaling, such as ipafricept (OMP-54F28), and PORCN inhibitors, such as ETC-159, LGK974 (WNT974), CGX1321 and RXC004 are targeted inhibitors of WNT/β-catenin signaling [30] (Fig. 1).

WNT/β-catenin signaling in health and disease
WNT/β-catenin is an evolutionally conserved singling [31] that is important in establishing and maintenance of cell-tocell adhesion [2].Dysregulation of WNT/β-catenin accounts for diseases like cancer.When WNT ligand is not present in the environment, β-catenin is assembled in related complex and low level of β-catenin is maintained within cytosol.β-catenin further undergoes phosphorylation and degradation.By contrast, bondage between WNT with related receptors prevent β-catenin degradation and allows its accumulation within cytosol and further translocation into nucleus for activating WNT-related transcription program [32].WNT/β-catenin maintains stemness in several epithelial tissues, which is important for development and regeneration of body organs [27].WNT/β-catenin signaling promotes self-renewal potential of hematopoietic stem cells [31], and its sustained activity in epidermal region expands stem cell compartment in the underlying dermis [33].Survival of immature CD4 + CD8 + T cells in thymus is also associated with β-catenin [34].The impact of WNT (WNT3a) on selfrenewal maintenance of CD8 + T cells, as occurring under normal conditions, represents implications of this signaling in vaccination or adoptive T cell therapy [31].
Increased β-catenin activity is a tumor hallmark [35], which is contributed to the initiation, progression, and invasion and metastasis of cancer [36].Hyperactive WNT/βcatenin signaling promotes aberrant cellular growth during cancer initiation [18].WNT/β-catenin is active in areas with vascular endothelial growth factor (VEGF)-related cold immunity [37], and the impact of β-catenin on P-glycoprotein is indicative of its involvement in multi-drug resistance [38,39].WNT/β-catenin reduces the expression of epithelial-related markers, such as E-cadherin [40], which is for acquisition of cancer stemness features.CSCs are PORCN + and provide WNT within their niches for tumor progressive purposes [41].

WNT/β-catenin impact on cellular immunity
Β-catenin activity mediates cooperation between tumor and stroma to promote cancer growth [42].Primary tumors show elevated expression of WNT/β-catenin in CD8 + T cells.Increased expression of genes related to the WNT/β-catenin pathway in lymphocytes is contributed to the apoptosis of mature T cells, and increased β-catenin signaling in tumor cells promotes T cell exhaustion [43].

WNT/β-catenin regulatory roles on PD-1/ PD-L1 and ICI responses
Increased PD-L1 expression is placed downstream to the β-catenin activity [38].Bondage of β-catenin/Tcf/Lef complex to the promoter of CD274 gene induces PD-L1 expression on tumor cells [57], and the impact of WNT/βcatenin on PD-L1 activation is indicative of the key role of this signaling in regulation of tumor immune landscape [58].Increased activity of WNT/β-catenin signaling is the underlying mechanism contributed to the development of non-inflamed TME and low ICI responses in highly mutated cancer type like NSCLC.In such cancer type, high tumor-mutational burden (TMB) is representative of low responses to ICI therapy [13].This is in contrast with the common belief that a tumor with higher somatic mutations generally shows higher responses to immunotherapy due to being more accessible to be killed by immune system [59].The high TMB in NSCLC is accompanied by lack of CD8 + T cell in TME and the resultant promotion of ICI resistance.This is due to the increased activity of WNT/β-catenin, which impairs CD8 + T cell infiltration into the tumor area [13].β-catenin activation is contributed to anti-PD-1 resistance in HCC [49].B-cell lymphoma 9 (BCL9) is the co-activator of β-catenin.Pharmacological blockade of β-catenin/BCL9 using desired peptides is reported to reduce the proportion of Tregs, increased tumoral infiltration of cytotoxic T lymphocytes and sensitized cancer cells to anti-PD-1 therapy [46].Lack of T cell genomic signature and T cell infiltrate due to the intrinsic tumor-mediated WNT/β-catenin activity is contributed to the anti-PD-L1 resistance of melanoma [4].There is a report of increased WNT/β-catenin in CD8 + T cells after anti-PD-1 therapy of primary sarcomas [43].Constitutive activation of WNT/β-catenin and further decreased expression of the chemokine CCL4 seemingly account for ineffective ICI responses [60].WNT/β-catenin mediates resistance to ICI therapy in part through blockade of cytokines contributed to the recruitment of immune cells.Targeting CTNNB1 using the nanoparticle drug product DCR-BCAT is attested to augmented T cell infiltration and increased tumor sensitivity to ICI therapy [61].

Combination of WNT/β-catenin inhibitors with anti-PD-1/PD-L1
Inhibitors of WNT/β-catenin can be developed to exert synergistic anti-tumor effects with ICIs in cancer immunotherapy [82].There is a report in HCC mice model indicating potent anti-tumor efficacy of nanoparticles constructed to simultaneously target hyperactive WNT/β-catenin and block endogenous PD-L1 [83].Takeuchi and colleagues attested a positive impact of WNT/β-catenin on ICI resistance in TMB high NSCLC, and the combination therapy with WNT/β-catenin blockade and anti-PD-1 better promoted anti-tumor immunity compared with either agent alone [13].Microsatellite stable (MSS) CRC shows dismal responses (0%) to ICI therapy.Combination of the PORCN inhibitor ETC-159 with the PD-1 inhibitor nivolumab reduced tumor volume in mice engrafted with MSS CRC.Combination therapy increased the fraction of effector CD4 + and CD8 + T cells and reduced Treg population, and augmented the antigen presentation profile represented by increased tumoral cell expression of major histocompatibility complex class II (MHC II) [84].Elevated activity of WNT ligand signaling is also responsible for failure of anti-PD-1 in melanoma.Suppression of WNT ligand increases the efficacy of anti-PD-1 in autochthonous animal tumor models through reduction of G-MDSC recruitment and reversion of DC tolerance.The higher suppressive impact of vantictumab or ipafricept over solo anti-PD-1 is reported in animal tumor model of melanoma, which is correlated with higher intra-tumoral infiltration of tumor-specific CD8 + T cells.DeVito and colleagues attested a positive link between anti-PD-1 resistance with increased WNT ligand signaling, which is indicative of the sensitivity of anti-PD-1 refractory melanoma to the WNT ligand blockade, as shown after application of ETC-159 [30].The efficacy of WNT974 plus the PD-1 inhibitor spartalizumab was evaluated in patients with advanced solid cancers.Treatment-related adverse events (TRAEs) were reported in 78% of patients, with hypothyroidism identified in 19% of cases.53% of patients who were refractory to prior anti-PD-1 showed stable disease, with uveal melanoma all cases (n = 5) represented stable disease.The outcomes are indicative of a presumable synergistic activity of the combined WNT pathway inhibition with ICI therapy against advanced solid cancers [22] (Table 1).In summary, it is rationale to assert that dysregulation of the WNT/β-catenin occurs in the context of human cancers and is associated with several cellular processes involved in tumor progression.Failure of anti-checkpoint therapy is a multi-mechanistic issue, among which the activity of WNT/β-catenin signaling has recently taken important consideration due to its critical association with cancer stemness.Tight interactions between WNT/β-catenin with different cells within tumor immune ecosystem, close interactions with PD-1/PD-L1 axis, and the promising outcomes from clinical trials targeting the two are all indicative of the application of combination therapies using WNT/β-catenin inhibitors with anti-PD-(L)1 in cancer immunotherapy, particularly in tumors with cold immunity and highly aggressive profile.However, there are points require attention when interpreting outcomes in patients under exposure to the combined WNT/β-catenin inhibitor/anti-PD-(L)1 therapy.First, interactions between WNT with complex receptors can activate signaling either dependent or independent on β-catenin, and a hallmark of a β-catenin-dependent pathway is its stability and nuclear translocation [86].Second, β-catenin transactivation can also occur independent on WNT [87], and Tcf1/Lef1 can also be activated by other transcription factors, such as ATF2 [88].Third, genotoxic agents can activate WNT/β-catenin independent on canonical Fzd/LRP receptor complex [89].Further studies are demanded for surveying other upstream mediators or inhibitors of β-catenin activity.5-carboxy-8-hydroxyquinoline (IOX1), for instance, is a histone demethylase inhibitor that suppresses Jumonji domain-containing 1A (JMJD1A) and its downstream β-catenin, and downregulates tumoral PD-L1, expressed secondary to the doxorubicin chemotherapy [38] (Fig. 4).The presence of WNT/β-catenin signaling in circulating extracellular vesicles (EVs) [90], and surface representation of PD-L1 by EVs secreted from tumor cells [91] are all indicative of a possibility for application of EVs in cancer immunotherapy targeting both signaling.A key virtue of such strategy is the tendency of EVs for their preferential attraction toward tumor tissue area due to expressing receptors related to that tumor type.Besides WNT/β-catenin, the activity of TGF-β signaling is also contributed to the stemness of tumor cells and cancer resistance to ICI therapy.Bispecific antibodies against TGF-β and PD-L1 are developed, and impressive responses are for PD-L1 high platinum refractory NSCLC patients [92].
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